Proteins are used as pharmaceuticals for the treatment of diseases in humans and other animals. About 50% of the top selling biopharmaceutical products in 2013 were therapeutic proteins, such as insulin and interferon (IFN-α-2a).
Many pharmaceutical proteins contain cysteine residues. In proteins with more than one cysteine residue, the thiol groups of the cysteine residues are usually covalently linked with other cysteine residues by specifically paired disulfide bonds. The therapeutically active form of a protein is usually a single disulfide species. While the native form of most proteins is a single disulfide species, some may cycle between a few disulfide states as part of their function, e.g., thioredoxin. In proteins with more than two cysteines, non-native disulfide species may be formed, which are typically misfolded. As the number of cysteines increases, the number of nonnative species can increase factorially.
Proteins may be made by recombinant DNA technology in host cells in which correct disulfide bond formation does not take place. For example, therapeutic proteins are typically produced using recombinant technology and a host expression system. E. coli based expression systems are widely used because of their simplicity, low cost, and relatively high protein yield. Overexpressed proteins accumulate in cytoplasm as inclusion bodies, which are biologically inactive.
A well-designed dissolution and refolding process is required to restore their native structures and biological activity. Generally, this refolding process is considerably slower for cysteine-containing proteins due to the formation of non-native disulfide bonds. The therapeutic proteins containing cysteine residues generally require the formation of proper disulfide bonds to maintain their native structures and stability. Large yield loss during the refolding step is a common problem and contributes significantly to the high production costs of therapeutic proteins. Refolding yield is usually low because of aggregation of the folding intermediates, the formation of non-native disulfide bonds, or stable, non-native tertiary structures.
This problem of yield loss during refolding is especially important for proteins with two or more disulfide bonds, such as proinsulin, interleukins, and growth factors. Unproductive aggregation reactions compete with slow productive disulfide bond formation and reshuffling reactions, resulting in significant yield loss. Although protein refolding processes have been revamped over the past decades with the introduction of on-line monitoring, folding additives, and on-column folding, the refolding procedures still requires optimization on a case-to-case basis. The molecular events and their affiliated kinetics contributing to the yield loss during refolding processes remain elusive for many systems. No refolding methods have emerged as the universal method. There remains a need to develop a rationally designed refolding procedure for increasing the yield of therapeutic proteins.
Insulin is the only therapeutic protein that is produced on the tons-per-year scale. In 2011, global insulin sales reached $16.7 billion, most of which was generated by three companies: Novo Nordisk (41%), Sanofi-Aventis (32%), and Eli Lilly (20%).6,7 While Novo Nordisk uses a yeast expression system to produce insulin, Sanofi-Aventis and Eli Lilly produce insulin using recombinant E. coli. Insulin is expressed as a precursor of proinsulin and stored in the E. coli cells as inclusion bodies. To produce insulin, the inclusion bodies must be isolated, denatured, sulfitolyzed, and refolded to form proinsulin-S-sulfonate (hPSS). During the in vitro folding of hPSS, a significant fraction of the folding intermediates aggregate through intermolecular disulfide bond formation, resulting in a yield loss of 40% or more. Of all the process steps for manufacturing insulin, the folding step has the lowest yield. Increasing in-vitro folding yield of proinsulin can significantly increase plant productivity, reduce raw materials and wastes, and reduce the cost of insulin produced from recombinant E. coli. 
There remains a need for improved refolding methods for therapeutic proteins, such as hPSS, that overcome the aforementioned deficiencies.